EP3620212A1 - Fermented violet extract, method for obtaining such an extract and use in cosmetics - Google Patents

Abstract

The invention relates to a fermented extract of an aerial part of violet obtained by a process comprising the preparation of a liquid extract by aqueous extraction of said aerial part; preparing a fermentation solution by mixing this liquid extract with a fermentable sugar; seeding this solution with a mixture of microorganisms containing at least one species of acetic bacteria and at least one species of yeast, this mixture being essentially free of lactic acid bacteria and lactose fermenting yeasts; incubation at a temperature less than or equal to 30 ° C for at least 10 days; and recovering the obtained liquid fermented extract. This fermented extract finds application as a cosmetic active principle for the care of the skin.

Description

The present invention is in the field of treatment of the skin, mucous membranes and / or hair of individuals, in particular the improvement of the cosmetic properties of the skin, mucous membranes and / or hair of individuals, and in particular of the fight against skin aging.

More particularly, the present invention relates to a fermented extract of an aerial part of violet, in particular of leaves, which is particularly suitable for use as a cosmetic active principle, in particular for combating skin aging. The invention also relates to a process for obtaining such a fermented extract, as well as a composition containing such an extract from fermented violet leaves, and a process for cosmetic treatment of the skin using such a composition. The invention further relates to such a fermented extract for its use for the treatment of acne and / or scars.

The violet, common name used to designate the plant genus Viola, is a plant in the Violaceae family .

Various species of the genus Viola have been used medicinally for decades to combat various health conditions. In particular, it has been identified and used in the medical field for the antifungal, antibacterial, analgesic, anti-inflammatory and diuretic effects of these species.

The document US 2015/0224158 describes in particular various medical applications of plant extracts, in particular violet leaves, obtained by fermentation of plants with kefir grains. Among such medical applications, mention may be made in particular of the use of plant extracts thus fermented as an antiseptic, antitussive, anti migraine, etc.

In general, fermentation technology consists in using microorganisms, more particularly bacteria and / or yeasts, which break down organic matter to form fermentation products of different structures and properties depending on the type of microorganisms used. These fermentation products can find application in many fields.

The present inventors are interested in the properties of fermented extracts of aerial parts, in particular leaves, violets, in particular of the species Viola alba (Besser, 1809), and in particular of the subspecies Viola alba subsp. dehnhardtii (Ten.) (W. Becker, 1902), commonly known as "Toulouse violet".

They discovered during their work that, quite surprisingly, an extract of aerial part, in particular of leaves, of violet having been subjected to fermentation by a particular ferment, exhibits properties which prove to be quite advantageous in the cosmetic field, and in particular in the field of the fight against skin aging, complex process resulting in the appearance of wrinkles, sagging skin and subcutaneous tissues, loss of elasticity of the skin , etc.

The present invention thus aims to provide a violet extract which can be used as a cosmetic active principle, in particular for topical use, and which is effective for the care, protection and / or treatment of the skin.

The invention aims in particular to ensure that this cosmetic active principle makes it possible to meet a major objective of the cosmetic field, namely to offer consumers a cosmetic composition making it possible to effectively prevent and / or slow down skin aging.

Thus, there is proposed according to the present invention a fermented extract of an aerial part of violet, usable in particular as a cosmetic active principle. In the present description is meant, conventionally in itself, by the term violet, a plant of the genus Viola.

• préparation d'un extrait liquide par extraction aqueuse de la partie aérienne de violette,
• préparation d'une solution dite de fermentation, par mélange de cet extrait liquide avec un sucre fermentescible,
• ensemencement de cette solution de fermentation avec un mélange de microorganismes contenant au moins une espèce de bactéries acétiques et au moins une espèce de levure, ce mélange étant essentiellement dépourvu de bactéries lactiques et de levures fermentant le lactose,
• incubation de la solution de fermentation ainsi ensemencée à une température inférieure ou égale à 30 °C, notamment comprise entre 25 et 30 °C, pendant au moins 10 jours, de préférence pendant au moins 20 jours,
• et récupération de l'extrait fermenté liquide ainsi obtenu.

This fermented extract of an aerial part of violet is obtained by a process for obtaining comprising steps of:

• preparation of a liquid extract by aqueous extraction of the aerial part of violet,
• preparation of a so-called fermentation solution, by mixing this liquid extract with a fermentable sugar,
• inoculation of this fermentation solution with a mixture of microorganisms containing at least one species of acetic bacteria and at least one species of yeast, this mixture being essentially devoid of lactic acid bacteria and lactose-fermenting yeasts,
• incubation of the fermentation solution thus seeded at a temperature less than or equal to 30 ° C, in particular between 25 and 30 ° C, for at least 10 days, preferably for at least 20 days,
• and recovering the liquid fermented extract thus obtained.

The mixture of microorganisms used according to the invention, containing at least one species of acetic bacteria and at least one species of yeast, will be designated in the present description, for convenience, by the term "ferment".

Acetic bacteria are defined here in a conventional manner in themselves, as bacteria whose aerobic metabolism produces acetic fermentation by redox. This metabolism is manifested specifically by a complete or partial conversion cycle of carbohydrates, primary alcohols, polyols or aldehydes, which results in particular from acetic acid.

• bactéries lactiques, c'est-à-dire de bactéries aptes à produire de l'acide lactique, notamment par fermentation des sucres, en particulier des bactéries de la famille des Lactobacillaceae, et notamment du genre Lactobacillus ;
• et de levures fermentant le lactose, telles que des levures de l'espèce Kluyveromyces marxianus.

According to the present invention, the ferment used is essentially devoid of, that is to say that it does not contain, or only in minute quantities, of:

• lactic acid bacteria, that is to say bacteria capable of producing lactic acid, in particular by fermentation of sugars, in particular bacteria of the Lactobacillaceae family , and in particular of the genus Lactobacillus ;
• and lactose fermenting yeasts, such as yeasts of the species Kluyveromyces marxianus.

The aerial part of the violet used for the preparation of the fermented extract according to the invention may consist of leaves, flowers, stems, or any mixture of these.

In particularly preferred embodiments of the invention, the method is implemented from an aerial part of violet containing violet leaves, and preferably essentially consisting of violet leaves. It is understood, by essentially consisting of violet leaves, that the aerial part selected according to the invention contains only violet leaves, to the exclusion of any other part of the plant, except in minimal quantities, that is to say say less than 1% by weight.

The present inventors have notably discovered that the fermentation of aqueous extracts of aerial part of violet, and very particularly of violet leaves, by such a ferment, proves to be much more effective than when it is carried out with kefir grains, such as proposed by the prior art. This performance is measured in particular in terms of speed and yield of fermentation per unit of ferment used.

The fermented extract obtained according to the invention has particularly advantageous properties for application in the cosmetic field, in particular for preventing or slowing down the effects of aging of the skin. For example, it has been discovered by the present inventors that when brought into contact with skin cells, the fermented violet extract according to the invention induces an increase in the production, by these cells, of one of the proteins known to present a skin anti-aging effect, collagen.

The fermented violet extract according to the invention can also meet one or more of the characteristics described below, used in isolation or in each of their technically operative combinations.

The process for obtaining the fermented extract according to the invention is preferably carried out from violet of the species Viola alba, and preferably from the subspecies Viola alba subsp. dehnhardtii. As explained above, this subspecies is commonly referred to as "Toulouse violet". The extracts fermented in accordance with the present invention from an aerial part, and very particularly from the leaves, of this particular plant, have a particularly significant anti-aging effect.

The step of aqueous extraction of an aerial part of violet can be carried out from any fresh part, for example from fresh leaves. It is preferably made from a dried part, in particular from dried leaves. It can also be carried out on the whole part or cut into pieces, as well as on a powder obtained by grinding.

Thus, the process for obtaining the fermented extract according to the invention may comprise an initial step of powdering the aerial part, in particular of the leaves, of violet.

The aqueous extraction step of the process for obtaining the fermented extract according to the invention can be carried out cold. It is preferably carried out hot.

In particular embodiments of the invention, the aqueous extraction is carried out at a temperature between 50 and 95 ° C, preferably between 80 and 95 ° C, for example at a temperature of 85 ° C ± 2 ° C or 90 ° C ± 2 ° C.

The aqueous extraction can be carried out by any conventional method in itself known to those skilled in the art, in particular by infusion, decoction, percolation or else maceration. It is preferably made by decoction.

The duration of the aqueous extraction is also preferably between 20 and 60 minutes, in particular between 20 and 30 minutes.

Preferably, the quantity of solid material (aerial part of violet) used is between 10 and 30 g per liter of water.

In particular embodiments of the process for obtaining of the fermented extract according to the invention, the aqueous extraction is carried out at 85 ° C ± 2 ° C for 20 to 30 minutes, from 10 to 30 g of powder per liter of water, and preferably by decoction .

At the end of the aqueous extraction step, the reaction medium is preferably subjected to filtration, so as to separate the materials insoluble in water from the liquid extract obtained.

The process for obtaining a liquid fermented extract according to the invention then comprises a step of preparing a fermentation solution. This preparation comprises the mixture, with the liquid extract obtained by aqueous extraction, of one or more fermentable sugars, that is to say sugar (s) which can be fermented by microorganisms, to form in particular the alcohol.

As examples of fermentable sugar which can be used according to the invention, mention may be made of glucose or sucrose. Sucrose, a disaccharide formed by the condensation of two oses, a glucose molecule and a fructose molecule, is particularly preferred in the context of the invention. Complex products containing one or more fermentable sugars, such as honey or Agave syrup, can also be used in the context of the invention.

The amount of fermentable sugar mixed with the liquid extract to form the fermentation solution is for example between 50 and 70 g / l.

• au moins une, de préférence plusieurs, espèces de bactéries acétiques, en particulier au moins une, de préférence plusieurs, espèces de bactéries du genre Acetobacter, par exemple Acetobacter sp, et/ou du genre Gluconobacter, par exemple Gluconobacter oxydans ;
• et au moins une, de préférence plusieurs, espèces de levure, en particulier au moins une, de préférence plusieurs, espèces de levures de la famille des Saccharomycetaceae, notamment du genre Saccharomyces, par exemple Saccharomyces cerevisiae, ou du genre Zygosaccharomyces, par exemple Zygosaccharomyces sp, et/ou de la famille des Schizosaccharomycetaceae, notamment du genre Schizosaccharomyces, par exemple Schizosaccharomyces pombe.

In particularly advantageous embodiments of the process for obtaining a fermented extract according to the invention, the mixture of microorganisms (ferment) used contains:

• at least one, preferably several, species of acetic bacteria, in particular at least one, preferably several, species of bacteria of the genus Acetobacter, for example Acetobacter sp, and / or of the genus Gluconobacter, for example Gluconobacter oxidans ;
• and at least one, preferably several, yeast species, in particular at least one, preferably several, yeast species of the family of Saccharomycetaceae, in particular of the genus Saccharomyces, for example Saccharomyces cerevisiae, or of the genus Zygosaccharomyces, for example Zygosaccharomyces sp, and / or of the family of Schizosaccharomycetaceae, in particular of the genus Schizosaccharomyces, for example Schizosaccharomyces pombe.

For sowing, different quantities of each of the microorganisms entering into the composition of the ferment can be used, in any weight ratio of one to the other.

The bacteria are preferably used in an amount of between 10 ² and 10 ⁸ CFU (Colony-forming Units) / ml of fermentation solution, preferably between 10 ⁴ and 10 ⁷ CFU / ml.

The yeasts are preferably used in an amount between 10 ² and 10 ⁷ CFU / ml of fermentation solution, preferably between 10 ³ and 10 ⁶ CFU / ml.

The microorganisms can be incorporated into the aqueous extract individually, or else in the form of a symbiotic ferment, that is to say a mixture of microorganisms which have naturally developed jointly in symbiosis, in particular during a culture of microorganisms. in the presence of an aqueous extract of the aerial part of violet and a fermentable sugar, for example for at least 15 days at a temperature of about 25 ° C.

In particular embodiments of the process for obtaining a fermented extract according to the invention, the mixture of microorganisms is supported on a solid support, preferably insoluble in water, in particular on a natural solid support produced by microorganisms, and more particularly by one or more of the microorganisms used in the composition of the ferment used according to the invention.

Preferably, the mixture of microorganisms is supported on a solid gelatinous support insoluble in water based on cellulose, hemicellulose or one of their derivatives, in particular based on hemicellulose bacterial, in particular produced by bacteria of the species Gluconobacter oxydans.

By way of example, the hemicellulose forming the solid support can correspond to the general formula:

The term cellulosane is used to denote this hemicellulose.

The incubation of the fermentation solution seeded with the mixture of microorganisms according to the invention is preferably carried out at a temperature between 25 and 30 ° C.

It is also preferably carried out for a period of between 20 and 30 days.

At the end of this incubation step, the liquid fermented extract obtained is recovered. This recovery can in particular be carried out by filtration, so as to eliminate the solid residues contained in the liquid medium. Filtration can in particular be carried out on a filter with pore diameters of 1 μm.

If necessary, glycerin can be added to the filtrate thus obtained.

In particular embodiments of the invention, the process for obtaining a liquid fermented extract according to the invention comprises a final stage of sterilization of the liquid fermented extract recovered at the end of the incubation, especially in moderate conditions, for example by tyndallization.

By way of example, the liquid fermented extract can be subjected to thermalization by batch heating at 70 ° C. for around thirty minutes, a few hours apart, and this three times. Such a treatment advantageously makes it possible to eliminate all the spores which may be contained in the liquid fermented extract.

The liquid fermented extract of an aerial part of violet according to the invention preferably has a pH less than or equal to 3. It also preferably has a Brix degree less than or equal to 2, in particular between 1 and 2. It it is preferably a fermented extract of violet leaves.

The process for obtaining a fermented extract of an aerial part of violet according to the invention can optionally comprise a final stage of lyophilization of the extract, to form a fermented extract in powder.

• préparation d'un extrait liquide par extraction aqueuse de la partie aérienne, notamment de feuilles, de violette,
• préparation d'une solution dite de fermentation par mélange de cet extrait liquide avec un sucre fermentescible,
• ensemencement de cette solution de fermentation avec un mélange de microorganismes contenant au moins une espèce de bactéries acétiques et au moins une espèce de levure, ce mélange étant essentiellement dépourvu de bactéries lactiques et de levures fermentant le lactose,
• incubation de la solution de fermentation ainsi ensemencée à une température inférieure ou égale à 30 °C pendant au moins 10 jours, de préférence pendant au moins 20 jours,
• et récupération de l'extrait fermenté liquide obtenu.

According to another aspect, the present invention relates to a process for obtaining a fermented extract of aerial part of violet according to the present invention. This process includes steps of:

• preparation of a liquid extract by aqueous extraction of the aerial part, in particular from leaves, violets,
• preparation of a so-called fermentation solution by mixing this liquid extract with a fermentable sugar,
• inoculation of this fermentation solution with a mixture of microorganisms containing at least one species of acetic bacteria and at least one species of yeast, this mixture being essentially devoid of lactic acid bacteria and lactose-fermenting yeasts,
• incubation of the fermentation solution thus sown at a temperature less than or equal to 30 ° C for at least 10 days, preferably for at least 20 days,
• and recovering the liquid fermented extract obtained.

This process can meet one or more of the characteristics described above with reference to the fermented extract of an aerial part of violet according to the present invention.

Another aspect of the present invention relates to a composition, in particular a cosmetic composition, in particular applicable topically to the skin, mucous membranes and / or hair of an individual. This composition contains a fermented extract of an aerial part, in particular of leaves, of violet according to the invention, meeting one or more of the characteristics described above, in a physiologically compatible vehicle.

The term “physiologically compatible vehicle” is intended to mean a vehicle suitable for use in contact with human and animal cells, in particular skin cells, in particular by topical application. Preferably, this vehicle has a pleasant odor, color and feel, and it does not generate unacceptable discomfort capable of diverting a user from the composition according to the invention.

The content of the fermented extract of an aerial part of violet according to the invention in the composition may in particular be between 0.1 and 10% by weight, relative to the total weight of the composition.

In addition to the fermented extract of an aerial part of violet according to the invention, as described above, the composition according to the invention may contain one or more additional active ingredient (s), in particular ingredients with beneficial properties for the skin, the effect of which can, if necessary, be exerted in synergy with that of the fermented violet extract according to the invention. By way of examples, mention may in particular be made of moisturizing agents, emollient agents, anti-wrinkle agents, anti-radical agents, anti-UV agents, etc.

The composition according to the invention can also comprise one or more conventional additives in themselves, in particular in the cosmetic field, for example one or more perfumes, stabilizing agent (s), preserving agent (s) ( s), emulsifier (s), etc.

This composition may be in any conventional form in itself, in particular for compositions for topical use, in particular for skin use, in the cosmetic field, in particular in the form of a cream, an ointment, a milk, d '' a lotion, gel, serum, ointment, etc.

Another aspect of the invention relates to a method of non-therapeutic cosmetic treatment of the skin, mucous membranes and / or hair of an individual, in particular human, this method comprising administration to this individual, in particular by application by topically on the skin, mucous membranes and / or hair of this individual, of a composition according to the invention, meeting one or more of the characteristics described above.

The cosmetic treatment method according to the invention comprises in particular the application of the composition according to the invention topically to a healthy area, not affected by a disease, in particular by an inflammatory disease, of the skin, the mucous membranes and / or the individual's hair.

The cosmetic treatment method according to the invention can in particular comprise the topical application, on the cutaneous surface of the concerned parts of the body of the individual in need thereof, for example on the skin of the face and the neck, of a determined amount of the composition according to the invention.

This application can for example be carried out once or twice a day, for example in the morning and in the evening, and for example for a period between 2 weeks and 2 months or more.

Applied topically to the skin, the composition according to the invention makes it possible, in particular, to prevent, or at least slow down, the appearance of skin signs of aging.

The cosmetic treatment process according to the invention may otherwise comprise the oral administration, to the individual concerned, of the composition according to the invention.

Another aspect of the invention relates to the use of a fermented extract of an aerial part, in particular of leaves, of violet according to the invention, meeting one or more of the above characteristics, for the non-therapeutic cosmetic treatment of the skin, mucous membranes and / or hair of an individual, in particular for combating skin aging.

More generally, the invention relates to the use of an extract of an aerial part, in particular of leaves, of violet, in particular of the species Viola alba, and preferably of the subspecies Viola alba subsp. dehnhardtii, for the non-therapeutic cosmetic treatment of the skin, mucous membranes and / or hair of an individual, in particular to combat skin aging

This use may in particular include the application of the violet extract topically, in particular on an area of the skin of the individual in need thereof, this area being healthy and not affected by a disease, in particular by a disease. inflammatory.

According to another aspect, the present invention relates to a fermented extract of an aerial part of violet according to the invention for its use for the therapeutic treatment of skin disorders, in particular with an inflammatory component, in particular for the treatment of acne and / or scars, in particular by topical application to the affected skin area of an individual, in particular human. This application advantageously takes advantage of the anti-inflammatory effect of the fermented extract according to the invention.

In particular, concerning the treatment of acne, it has been discovered by the present inventors that, quite surprisingly, the fermented extract of an aerial part of violet according to the invention has the capacity to slow down the appearance on the skin of biofilms of the bacteria Propionibacterium acnes and Staphylococcus epidermitis, which are involved in skin conditions such as acne ( Burkart and Burkart, 2003, Int. J. Dermatol. 42: 925-927 ), and to block the maturation of such biofilms. Such an action of the fermented extract according to the invention, which has the effect of promoting the disappearance of acne lesions in acne-prone skin and oily skin prone to acne, seems to be due to a deactivation of the quorum sensing, intercellular communication system of these bacteria.

In an entirely advantageous manner, the fermented extract according to the invention does not furthermore induce intolerance, allergy or aggressive side effects in contact with the skin.

• la figure 1 représente des profils obtenus par analyse HPLC respectivement pour une solution de fermentation (non ensemencée) obtenue en produit intermédiaire lors de la mise en oeuvre d'un procédé d'obtention d'un extrait fermenté de feuilles de Viola alba subsp. dehnhardtii selon l'invention (ligne avant), et pour un extrait fermenté de feuilles de Viola alba subsp. dehnhardtii selon l'invention, obtenu après fermentation de ladite solution de fermentation (ligne arrière), montrant uniquement les pics dont l'aire brute diffère entre les deux solutions ci-avant ;
• la figure 2 représente un graphe montrant le pourcentage d'augmentation de la quantité de collagène dans la matrice cellulaire de cellules de fibroblastes humains, induite par un extrait fermenté de feuilles de Viola alba subsp. dehnhardtii selon l'invention à différentes concentrations dans le milieu de culture des cellules, et par le facteur de croissance TGF-β à titre de contrôle positif, ce pourcentage étant calculé par rapport au milieu de culture non additionné ;
• la figure 3 montre des photographies de puits obtenus à l'issue de la mise en oeuvre du test Biofilm Ring Test® pour un témoin (« T ») et pour des compositions contenant différentes concentrations d'un extrait fermenté selon l'invention (« S2% » et « S4% »), vis-à-vis de bactéries respectivement Propionibacterium acnes (a/) et Staphylococcus epidermitis (b/) après des temps d'incubation respectivement de 0h, 2h, 4h, 6h et 24h ;
• la figure 4 montre un graphe en barres représentant le pourcentage de présence de biofilm, calculé par le test Biofilm Ring Test®, pour un témoin (« T ») et pour des compositions contenant différentes concentrations d'un extrait fermenté selon l'invention (« S2% » et « S4% »), vis-à-vis de la bactérie Propionibacterium acnes après un temps d'incubation de 24h ;
• et la figure 5 montre un graphe en barres représentant le pourcentage de présence de biofilm, calculé par le test Biofilm Ring Test®, pour un témoin (« T ») et pour des compositions contenant différentes concentrations d'un extrait fermenté selon l'invention (« S2% » et « S4% »), vis-à-vis de la bactérie Staphylococcus epidermitis après un temps d'incubation de 24h.

The characteristics and advantages of the invention will appear more clearly in the light of the examples of implementation below, provided for illustrative purposes only and in no way limit the invention, with the support of figures 1 and 5 , in which :

• the figure 1 represents profiles obtained by HPLC analysis respectively for a fermentation solution (not seeded) obtained as an intermediate product during the implementation of a process for obtaining a fermented extract of leaves of Viola alba subsp. dehnhardtii according to the invention (front line), and for a fermented extract of leaves of Viola alba subsp. dehnhardtii according to the invention, obtained after fermentation of said fermentation solution (rear line), showing only the peaks whose gross area differs between the two solutions above;
• the figure 2 represents a graph showing the percentage increase in the amount of collagen in the cellular matrix of human fibroblast cells, induced by a fermented extract of leaves of Viola alba subsp. dehnhardtii according to the invention at different concentrations in the cell culture medium, and by the growth factor TGF-β as a positive control, this percentage being calculated relative to the culture medium not added;
• the figure 3 shows photographs of wells obtained at the end of the implementation of the Biofilm Ring Test® test for a control (“T”) and for compositions containing different concentrations of a fermented extract according to the invention (“S2%” and “S4%”), with respect to bacteria respectively Propionibacterium acnes (a /) and Staphylococcus epidermitis (b /) after incubation times of 0h, 2h, 4h, 6h and 24h respectively;
• the figure 4 shows a bar graph representing the percentage presence of biofilm, calculated by the Biofilm Ring Test® test, for a control ("T") and for compositions containing different concentrations of a fermented extract according to the invention ("S2%" and "S4%"), vis-à-vis the bacteria Propionibacterium acnes after an incubation time of 24 hours;
• and the figure 5 shows a bar graph representing the percentage of presence of biofilm, calculated by the Biofilm Ring Test® test, for a control ("T") and for compositions containing different concentrations of a fermented extract according to the invention ("S2% "And" S4% "), vis-à-vis the bacteria Staphylococcus epidermitis after an incubation time of 24 hours.

A / Fermented extract of leaves of Viola alba subsp. dehnhardtii

The process for obtaining according to the following invention is implemented.

Leaves of the species Viola alba subsp. dehnhardtii are dried, then subjected to aqueous extraction by decoction.

For this purpose, the dried leaves are incorporated into 400 l of demineralized water at room temperature, at a rate of 20 g of leaves per liter of water. The aqueous medium is heated to 85 ° C ± 2 ° C, and maintained at this temperature for 60 min, until an optical density greater than 20 to 280 nm is obtained.

The aqueous medium is then filtered to remove solid residues.

A fermentation solution is prepared from the filtrate thus obtained.

For this, sucrose is added to the filtrate at a concentration of 70 g / l. The medium is stirred until the sucrose is completely dissolved, then filtered to remove the remaining solid impurities.

The fermentation solution thus obtained is seeded with a mixture of microorganisms containing bacteria of the species Acetobacter sp and Gluconobacter oxydans, and yeasts of the species Saccharomyces cerevisiae, Zygosaccharomyces sp and Schizosaccharomyces pombe, this mixture being essentially free of lactic acid bacteria and lactose fermenting yeasts.

The microorganisms are preferably trapped in a solid support insoluble in water, preferably based on hemicellulose.

Each microorganism is used at a rate of approximately 10 ⁶ CFU / ml of fermentation solution.

Preferably, a symbiotic ferment is used which has been obtained by seeding a fermentation solution containing an aqueous extract of leaves of the species Viola alba subsp. dehnhardtii and sucrose, such as the fermentation solution described above, with a mixture containing a large number of different microorganisms, and incubation at approximately 25 ° C for 15 days. After 15 days of incubation, a polymer product, cellulosane, was formed in the solution, which traps a particular mixture of microorganisms, containing bacteria of the species Acetobacter sp and Gluconobacter oxydans and yeasts of the species Saccharomyces cerevisiae, Zygosaccharomyces sp and Schizosaccharomyces pombe, this mixture being essentially devoid of lactic acid bacteria and lactose-fermenting yeasts. After filtration, this symbiotic ferment is recovered on a solid support.

20 kg of this ferment are incorporated into the fermentation solution.

The medium thus obtained is then incubated under static conditions at 28 ° C (± 2 ° C) for 30 days.

A Brix degree less than 2 and a pH less than or equal to 3 is then obtained for the reaction medium.

This medium is subjected to filtration so as to separate the ferment on solid support from the liquid fermented extract, first on a first filter with a pore diameter of 1 μm, then on a second filter with a pore diameter of 0, 2 µm.

The liquid fermented extract thus isolated is subjected to a treatment by tyndallization, by repeating the following cycle 3 times: heating at 70 ° C. for 20 min, cooling at room temperature for 24 h.

The extract of fermented violet leaves then obtained is stored protected from light.

This extract of fermented violet leaves, obtained in accordance with the present invention, is analyzed by high performance liquid chromatography (HPLC). By way of comparison, the fermentation solution formed during the above production process is also analyzed, not seeded with the ferment.

The HPLC analysis is carried out on an Agilent Poroshell 120 SB-C18 column, with injection of 10 μl of each sample (having been previously concentrated 3 times by vacuum evaporation), at a temperature of 30 ° C. and a flow rate of 0 , 8 ml / min, with detection at 210 nm. Eluent A (water and 7.5 mM formic acid) and eluent B (acetonitrile) are used in gradient mode, as follows:
0 min A 95% B 5%; 3 min A 95% B 5%,; 10 min A 92% B 8%; 12 min A 90% B 10%; 17 min At 87% B13%; 20 min A 80% B 20%; 27 min A 70% B 30%; 30 min A 50% B 50%; 31 min At 0% B100%; 34 min A 0% B 100%; 35 min A 95% B 5%; 38 min A 95% B 5%.

The results obtained are shown on the figure 1 . In this figure, the front line shows the results obtained for the liquid solution before fermentation, and the rear line shows the results obtained for the liquid solution after fermentation.

There is clearly a significant change in the composition of the solution due to fermentation.

B / Fermented extracts of leaves of other Viola species

• Viola tricolor (communément nommée pensée sauvage),
• Viola odorata (communément nommée violette odorante).

The process described in example A / above is implemented from leaves of other species of violets:

• Viola tricolor (commonly known as wild pansy),
• Viola odorata (commonly known as fragrant violet).

For each of these species, a fermented extract is obtained in accordance with the invention.

C / Activation of collagen synthesis by human fibroblasts

The leaf extract of Viola alba subsp. fermented dehnhardtii obtained in Example A / is tested for its capacity to activate the synthesis of collagen by human fibroblasts.

For this purpose, primary human fibroblast cells are cultured in a suitable culture medium (DMEM medium 4.5 g / l glucose, 2 mM L-glutamine, 10% heat-inactivated fetal calf serum (SVF) , 50 IU / ml of penicillin, 50 μg / l of streptomycin), in the presence of different concentrations of the fermented extract according to the invention (5000 μg / ml, 1000 μg / ml, 500 μg / ml). The culture is carried out at 37 ° C. under a 5% CO ₂ atmosphere.

As a negative control, the effect of the culture medium alone, that is to say not added with the fermented extract according to the invention, at 1% of FCS, is tested. As a positive control, the growth factor TGF-β is tested at 10 ng / ml in the culture medium at 1% FCS.

For each sample tested, the amount of collagen present in the cell matrix and in the culture medium is determined after 48 h of culture, using Sirius red dye (Direct Red 80), which has a particular affinity for the triple structure. helix (Gly-XY) _n of native collagen. The total amount of protein present is also measured by the Bradford test.

For this purpose, after 48 h of culture, the culture medium is removed and the cell layer is rinsed with 500 μl of protease inhibitor cocktail. The cell layer is then recovered by scraping in 500 μl of protease inhibitor cocktail, then the culture wells are rinsed with 500 μl of the protease inhibitor cocktail. The two volumes, which constitute the extracellular matrix, are combined and treated by ultrasound for 40 s.

Collagen is complexed with the red dye Sirius. After centrifugation and washing with 0.1 M hydrochloric acid, the pellet is dissolved in a 0.5 M sodium hydroxide solution. The absorbance of the dye-collagen complex is measured at 540 nm. A calibration range is established between 0 and 50 µg per collagen tube.

The total quantity of proteins present is also measured by the Bradford test, according to the protocol described in the publication of Bradford et al., 1976, Anal. Biochem. 72: 248-54 , against a calibration range established from a solution of BSA (bovine serum albumin) at 400 μg / ml in PBS (phosphate buffered saline).

For each sample, the weight ratio of collagen / proteins measured by the Bradford test (in the cell matrix and in the culture medium) is calculated, as well as the% increase in the production of collagen by the cells, compared to the negative control.

The results obtained are shown in Table 1 below. <u> Table 1 </u> - effect of a fermented extract according to the invention on the production of collagen by human fibroblasts Bradford collagen / protein ratio % increase in collagen production compared to negative control Cellular matrix Culture centre Cellular matrix Culture centre Fermented extract according to the invention 5000 μg / ml 0.198 0.185 29 0 Fermented extract according to the invention 1000 μg / ml 0.212 0.209 38 13 Fermented extract according to the invention 500 μg / ml 0.145 0.172 3 0 Positive control (TGF-β 10 ng / ml) 0.233 0.168 51 0 Negative control (culture medium) 0.154 0.185 72 0

The figure 2 represents a graph showing the percentage increase in collagen production by human fibroblast cells, induced by the fermented extract according to the invention at different concentrations in the cell culture medium, and by the growth factor TGF- β, relative to the culture medium not added.

It is clear from the results obtained that the fermented extract of leaves of Viola alba subsp. dehnhardtii according to the invention significantly increases the production of collagen by human fibroblasts, from the concentration of 1000 μg / ml in the cell culture medium.

These results demonstrate the performance of the fermented extract according to the invention for combating skin aging.

D / Action against biofilm formation by Propionibacterium acnes and by Staphylococcus epidermitis

The leaf extract of Viola alba subsp. fermented dehnhardtii obtained in Example A / is tested for its capacity to fight against biofilms of Propionibacterium acnes and against biofilms of Staphylococcus epidermitis.

The aim of this study is to determine whether the ability of these bacteria to establish a biofilm, involved in acne, can be slowed down, and the maturation of this biofilm blocked, by the fermented extract according to the invention. For this purpose, the Biofilm Ring Test® method from Biofilm Control® is used. This method makes it possible to determine an index, called BFI (for the English BioFilm Index), which is inversely proportional to the level of production of biofilm ( Dagnelie et al., 2018, Annales de Dermatologie et de Vénéréologie 145 (12): S123 ).

In these experiments, the percentage of presence of the biofilm is determined initially (at 0 h) and after respectively 2 h, 4 h, 6 h and 24 h of incubation of the bacteria with the extract according to the invention.

The organisms used in this study are Propionibacterium acnes KPA 171202 and Staphylococcus epidermidis ATCC 12228.

The protocols used are as follows.

Regarding Propionibacterium acnes, a liquid aliquot of bacteria is prepared according to the slightly modified method of Hayes and Markovic ( Hayes and Markovic, 2002, Food Chem Toxicol. 40: 535-543 ). Propionibacterium acnes cells are incubated in a medium for heart and brain perfusion (BHI) with 1% glucose for 48 h at 37 ° C and under anaerobic conditions. The medium is diluted to obtain approximately 1.0 × 10 ⁸ CFU / ml.

Staphylococcus epidermidis cells are incubated in trypticase soy broth (TSB) for 24 h at 37 ° C. under aerobic conditions, then the medium is adjusted to obtain approximately 1.0 × 10 ⁸ CFU / ml.

The following compositions are prepared:
Control: 10 ml test tubes containing 3 ml of nutritive yeast glucose broth (NYG) for Propionibacterium acnes, or 3 ml of nutrient broth for Staphylococcus epidermidis, were autoclaved for 12 h at 150 ° C. The medium in each test tube was cooled and then inoculated with 50 μl of the bacterial suspension of Propionibacterium acnes or of Staphylococcus epidermidis containing 1x10 ⁸ cells / ml.

Compositions based on extract according to the invention: the extract according to the invention is added, respectively at 2% v / v (composition S2%) and at 4% v / v (composition S4%), in some of the test tubes above.

The principle of the Biofilm Ring Test® method is described by Chavant et al. 1997, J Med Microbiol 60: 300-306 . It is based on the addition to the bacterial solution, containing if necessary the extract to be tested, of magnetic beads of 5 or 6 μm in diameter. The mixture of magnetic beads and bacterial solution is seeded in wells, then incubated for different durations between 0h and 24h. The aerobic or anaerobic conditions necessary for the development of bacterial biofilms for each of the strains tested are respected. After the incubation period in the wells, a contrast medium is added to allow reading in the special Biofilm Reader®. After the first reading, the data collected by the reader is sent to the computer, interpreted and stored by the special Biofilm Control Elements® software. The wells are then inserted into the magnetizer and magnetized for 1 min. The beads which have not been retained in the biofilm possibly formed are attracted towards the center of the well, forming a red circle there. As the biofilm is formed, the particles are immobilized there throughout the volume of the well and the red circle disappears. After the magnetization, a second reading is carried out to allow the detection of the magnetic beads deposited on the base of the well. The data collected after this second reading is also transmitted, interpreted and stored on the computer. From the comparison of the images obtained after the first and second readings for each well, a value called the Biofilm Formation Index (BFI) is calculated, as well as the percentage of presence of biofilm in the well.

The figure 3 shows photographs of the wells after different incubation times for the control ("T") and for the two concentrations of fermented extract according to the invention ("S2%" and "S4%") for the different incubation times , in a / for Propionibacterium acnes and in b / for Staphylococcus epidermitis.

As we can observe, concerning Propionibacterium acnes, at 0h all the samples show an absence of biofilm (central ring clearly visible in the photographs); from 2 hours of incubation, the control produces biofilm in quantity and this up to 24 hours of incubation (the central ring is no longer visible, testifying to the fact that the particles have been immobilized in the biofilm formed). For the samples containing the extract according to the invention (S2% and S4%), an absence of biofilm is observed at all the incubation times tested.

Concerning Staphylococcus epidermitis, from 0h to 4h all the samples show an absence of biofilm; from 6 hours of incubation the control produces biofilm in quantity, and this up to 24 hours. For the samples containing the extract according to the invention (S2% and S4%), the absence of biofilm at all incubation times tested.

For all samples, the percentage of biofilm presence after 24 hours of incubation is calculated. The results are shown on the figure 4 for Propionibacterium acnes and on the figure 5 for Staphylococcus epidermitis.

As can be observed, the witness shows that Propionibacterium acnes is 100% biofilmogenic, that is to say that the quorum sensing is maximum and produces biofilm. The sample according to the invention S2% shows a reduction in biofilm formation by 55%, therefore by more than half compared to the control. The sample according to the invention S4% shows a reduction in the formation of biofilm by 85%, that is to say that the quorum sensing could not develop the maturation of the biofilm.

Concerning Staphylococcus epidermitis, here again the witness shows that Staphylococcus epidermitis is 100% biofilmogenic, that is to say that the quorum sensing is maximum and produces biofilm. The sample according to the invention S2% shows a reduction in the formation of biofilm by 46% compared to the control. The sample according to the invention S4% shows a reduction in biofilm formation of 72%, that is to say that the quorum sensing could not develop the maturation of the biofilm.

All of these results clearly demonstrate the anti-biofilm efficacy of the fermented extract according to the invention on the two strains of bacteria tested, which are associated with acne disorders. By preventing the production of biofilm, the extract according to the invention prevents the formation of sessile bacteria, and both Propionibacterium acnes and Staphylococcus epidermitis remain in symbiosis with the keratinocytes of the skin and retain their commensal activity for protecting the skin barrier.

Claims (12)

Fermented extract of an aerial part of violet, obtained by a process for obtaining comprising steps of: - preparation of a liquid extract by aqueous extraction of said aerial part of violet, - preparation of a so-called fermentation solution by mixing said liquid extract with a fermentable sugar, characterized in that said method comprises steps of: inoculation of said fermentation solution with a mixture of microorganisms containing at least one species of acetic bacteria and at least one species of yeast, said mixture being essentially free of lactic acid bacteria and yeasts which ferment lactose, - incubation at a temperature less than or equal to 30 ° C for at least 10 days, - and recovery of the liquid fermented extract obtained. Extract according to claim 1, according to which said method is carried out from violet of the species Viola alba, preferably of the subspecies Viola alba subsp. dehnhardtii. Extract according to any one of claims 1 to 2, according to which the said method is carried out from an aerial part of violet containing violet leaves, and preferably essentially consisting of violet leaves. Extract according to any one of claims 1 to 3, according to which, in said production process, the aqueous extraction is carried out at a temperature between 50 and 95 ° C, preferably between 80 and 95 ° C. Extract according to any one of claims 1 to 4, according to which, in said production process, said fermentable sugar is sucrose. Extract according to any one of claims 1 to 5, according to which, in said production process, said mixture of microorganisms contains: - at least one species of bacteria of the genus Acetobacter or of the genus Gluconobacter, - And / or at least one yeast from the Saccharomycetaceae family or from the Schizosaccharomycetaceae family . Extract according to any one of claims 1 to 6, according to which, in said production process, said mixture of microorganisms is supported on a solid support, preferably on a solid support based on hemicellulose. Extract according to any one of claims 1 to 7, having a pH less than or equal to 3 and a Brix degree less than or equal to 2. Process for obtaining a fermented extract of an aerial part of violet according to any one of Claims 1 to 8, characterized in that it comprises the steps of: - preparation of a liquid extract by aqueous extraction of said aerial homeland of violet, - preparation of a so-called fermentation solution by mixing said liquid extract with a fermentable sugar, inoculation of said fermentation solution with a mixture of microorganisms containing at least one species of acetic bacteria and at least one species of yeast, said mixture being essentially free of lactic acid bacteria and yeasts which ferment lactose, - incubation at a temperature less than or equal to 30 ° C for at least 10 days, - and recovery of the liquid fermented extract obtained. Composition containing a fermented extract of an aerial part of violet according to any one of Claims 1 to 8, in a physiologically compatible vehicle. Method of non-therapeutic cosmetic treatment of the skin, mucous membranes and / or hair of an individual, characterized in that it comprises the administration to said individual of a composition according to claim 10. Fermented extract of an aerial part of violet according to any one of claims 1 to 8, for its use for the treatment of acne and / or scars.

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